The main goal was to test therapeutic efficacy and potential toxicity of TFEB activation in Pompe muscle cells. We have tested the effect of TFEB in GAA-/- and muscle-specific autophagy-deficient GAA-/- mice, the two models that were previously developed by our group. In addition, we have used two newly developed Pompe disease models an in virto myoblast cell lines and an in vivo GAA-/- model, in which autophagosomes are labeled with GFP- LC3 (GFP-LC3:GAA-/-). The use of multiple models enabled us not only to evaluate the effect of TFEB on lysosomal and autophagic pathologies, but also to examine the role of autophagy in TFEB-mediated exocytosis. We have shown that TFEB efficiently triggers lysosomal exocytosis and promotes cellular clearance in all tested PD model systems muscle cells, isolated live muscle fibers, and the whole muscle of PD mice. In all systems, the number of large glycogen-filled lysosomes and the amount of accumulated glycogen were significantly reduced, thus providing strong evidence of TFEBs potential as a therapeutic target in Pompe disease. Immortalized murine myoblasts derived from GAA-/ mice, unlike fibroblasts, accumulate lysosomal glycogen and, therefore, represent an excellent system to study cellular clearance. The simplicity of detecting hugely expanded lysosomes and glycogen in Pompe disease myotubes makes this model particularly attractive. The effects of TFEB were easily monitored and evident within days. Furthermore, the cell system allowed us to expose a potential problem with TFEB overexpression: a constitutively active form of TFEB, in which S211 is mutated to alanine, invariably resulted in gross morphological changes of myotubes, perhaps because of massive expulsion of the lysosomes. Analysis of TFEB-transfected live fibers (derived from different GAA-/ mouse strains) by time-lapse microscopy demonstrated that TFEB significantly relieved autophagic buildup by stimulating the formation and secretion of autophagolysosomes - a product of lysosomal-autophagosomal fusion. Our data suggest that lysosomal exocytosis may in fact not be a purely lysosomal event, but rather a process involving autophagy and secretion of autophagolysosomes. Indeed, the effects of TFEB on lysosomal velocity and clearance were much attenuated in autophagy-deficient PD mice, indicating that functional autophagy enables TFEB to exert its full effect. Potential applications of our newly developed models, PD myotubes and GFP-LC3:GAA -/- mice, extend beyond testing the effects of TFEB. For example, an in vitro PD model is much needed for large-scale drug testing. In recent years, several attempts have been made to develop such a model. Our new conditionally immortalized Pompe disease muscle cell lines have several advantages compared to previous models - they are highly myogenic, derived from individual clones, and, most importantly, they maintain the capacity to differentiate into multinucleated myotubes when the immortalizing gene is inactivated. The newly developed cell lines replicate lysosomal pathology but fail to recapitulate autophagic buildup (this failure is common to all in vitro PD models). A possible explanation is that the survival of cell cultures requires a nutrient-rich medium, which keeps autophagic activity at low levels. In addition, the survival time of the myotubes in culture may be shorter than the time needed for the accumulation of autophagosomes. Indeed, we observed massive accumulation of autophagosomes in long-term primary myotubes. We are planning to find a way to trick these cells into displaying the autophagic accumulation that is so striking in Pompe muscle fibers. In the interim, the newly generated GFP-LC3:GAA-/- mouse model fills the void by providing an excellent tool to address the role of autophagy. The autophagic buildup in muscles from these mice can be seen in live unprocessed fibers, in muscle bundles, and even in muscle tissues of a living mouse. In addition to using this model to study the role of autophagy in TFEB-mediated exocytosis, we have utilized GFP-LC3:GAA-/- mice to address intracellular trafficking of recombinant human GAA (rhGAA) and possible mechanisms of autophagic buildup. We have now direct evidence that the bulk of the administered recombinant enzyme ends up not in lysosomes but in autophagosomes within the area of buildup, as we suggested previously. As for the possible mechanisms of autophagic accumulation, we have found 1) an absence of fusion between autophagosomes and lysosomes in the buildup area and 2) strikingly low levels of LAMP expression in this region, suggesting inefficient local lysosomal biogenesis. A combination of these factors may underlie the buildup process, which appears to begin with the inability of a subset of lysosomes in the core of muscle fiber to fuse with autophagosomes, perhaps because of lysosomal rupture during muscle contractions. It is possible that this fusion defect and the paucity of the newly formed lysosomes in the autophagic area create conditions that favor expansion of the autophagic buildup. With the capacity to resolve both of these deficits by promoting lysosomal-autophagosomal fusion and biogenesis, TFEB is uniquely suited to address autophagic and lysosomal pathologies in Pompe disease. This work is done in collaboration with Dr. Rosa Puertollano (Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) and Andrea Ballabio (Telethon Institute of genetics and Medicine (TIGEM), Naples, Italy). In yet another approach to rendering Pompe skeletal muscle fibers responsive to therapy, we have made transgenic Pompe mice, which express myosin-encoded miR-499 under the control of the MCK promoter. The reprogramming of therapy resistant fast myofibers to a slower myofiber type (amenable to therapy) in MCK-miR-499:WT mice is achieved by a change in the contractile properties. We have found that these mice clear glycogen more efficiently on enzyme replacement therapy compared to GAA-/- mice, thus emphasizing potential benefits of endurance training as an adjunctive therapy to ERT. In addition, we plan to embark on a joint project with Dr. Vittorio Sartorelli. The project is designed to examine the role of autophagy in muscle development. To this end, we have generated a mouse strain, in which a critical autophagic gene, Atg 7, is inactivated in muscle stem cells under the control of Pax7 promoter. We have continued to analyze muscle biopsies from Pompe patients who receive enzyme replacement therapy. In a study of single muscle fibers from infants with Pompe disease we have noted that the dominant pathology is the presence of huge, glycogen-filled lysosome but the autophagic buildup, which is so prominent in the milder adult form of the disease, is negligible. The data point to the differences in the pathogenesis of Pompe disease in infants and adults, with possible implications for the design of therapy. However, in infants on ERT, as the glycogen-filled lysosomes are shrunk, autophagic buildup becomes visible. Until now we have had access to muscle biopsies from untreated late-onset patients and from infants who received the enzyme for 6 months. We are now conducting a long-term study to address the fate of the autophagic buildup after prolonged therapy, and to establish a link between the extent of autophagic accumulation and the clinical response to therapy.